61 18
J . Am. Chem. SOC.1985, 107, 6118-6120 metastable excimer state. In fluid media, the pyrene excimer takes a sandwichlike configuration. In the y-CDx cavity, however, the sterically restricted environment may not allow formation of a symmetric sandwich excimer. An asymmetrically twisted configuration seems to be taken under the conditions. In the case of P(3)P, an alkyl chain, which links two pyrenyl groups, seems to prohibit the configurational change of P(3)P included in the y-CDx cavity. This may cause the smaller gemvalue and the absence of the rise of the intramolecular excimer fluorescence. No wavelength dependence of the gemvalue over the whole excimer region was observed for the P(3)P-yCDx system, suggesting only one fluorescent state which shows CPF. As Figure 1 shows, however, the gemvalue for the pyrene-y-CDx system decreases at longer wavelength. There may be two fluorescent states, one is the excimer state and another is the dimer excited state, whose configuration is almost the same as that of the ground-state dimer of pyrene in the y-CDx cavity. The rise of the pyrene excimer fluorescence observed in the decay curve may be ascribed to the dynamic formation of the excimer state in the y-CDx cavity which seems to be a considerably tight environment for two pyrene molecules.
L
Wavelength, nm
Figure 1. Fluorescence (bottom) and CPF (top) spectra of pyrene (2 10" M) in aqueous y-CDx ( 1 X lo-* M) solution a t 25 "C.
X
toluene and dimethylformamide.22 This also suggests that the large gemin the present system is neither due to an artifact caused by a linear polarization nor due to an electronic perturbation by the y-CDx cavity on an achiral excimer. The electronically induced C P F has been reported for the systems of an achiral fluorescer (fluorescein) in a chiral solvent ( ~ h e n e t y l a m i n eand )~~ a 1: 1 inclusion complex of fluorescein and B - C D X . ~The ~ lgeml values for the electronically induced CPF, however, are significantly smaller (10-4-10-3) than that for the present system (0.00714.012). Consequently, it can be concluded that the pyrene molecules in the y-CDx cavity form the excimer having an asymmetrically twisted configuration. The intramolecular excimer systems can be regarded as the model guests for studing the 2.1 = guest:CDx We used 1,3-bis(1-pyreny1)propane(P(3)P). Since P(3)P is completely insoluble in water, a dilute aqueous dispersion M) shows only excimerlike fluorescence centered at 478 nm due to the P(3)P aggregates, which shows a multiexponential fluorescence decay.2s In the presence of y-CDx ( M), however, the fluorescence from the locally excited state of P(3)P was observed along with the intramolecular excimer fluorescence with a maximum intensity at 490 nm. The fluorescence decay of the P(3)P excimer was ~ 159 ns) in the presence of y-CDx and single exponential ( T = no rise of the excimer was observed, suggesting that the intramolecular dimer state is formed in the y-CDx cavity in the ground state. A relatively weak C P F signal was observed for the P(3)P-y-CDx system, gembeing ca. +0.003 at the excimer region. Recently, an induced circular dichroism (ICD) of the pyrene dimer formed in the y-CDx cavity has been reported.18 A very at the small absorption dissymmetry factor of ICD (ca. 6 X 'La band)29indicates a weak asymmetric nature of the pyrene dimer in the y-CDx cavity in the ground state. Upon excitation of a pyrene molecule, the reorientation of two pyrene molecules may occur in the y-CDx cavity to form thermodynamically (21) Brittain, H.; Ambrozich, D. L.; Saburi, M.; Fendler, J. H. J . Am. Chem. Soc. 1980, 102, 6372. (22) Egusa, S.; Sisido, M.; Imanishi, Y . Macromolecules 1985, 18, 882. (23) Brittain, H. G.: Richardson, F. S. J . Phys. Chem. 1976, 80, 2590. (24) Brittain, H . G. Chem. Phys. Lett. 1981, 83, 161. (25) Turro, N . J.; Okubo, T.; Weed, G. C. Photochem. Photobiol. 1982, 35, 325. (26) Emert, J.; Kodali, D.: Catena, R . J . Chem. SOC.,Chem. Commun. 1981, 758. (27) Arad-Yellin, R.; Eaton, D. F. J . Phys. Chem. 1983, 87, 5051. (28) Kano, K. Ueno, Y . ; Umakoshi, K.; Hashimoto, S.; Ishibashi, T.; Ogawa, T. J . Phys. Chem. 1984, 88, 5087. (29) The gab( = A e / c ) value was calculated from the ICD and absorption spectra were reported in ref 18 by the present authors.
0002-7863/85/1507-6118$01.50/0
Use of Differential Second-Derivative UV and FTIR Spectroscopy in Structural Studies of Multichromophoric Compounds Gregory L. Verdine and Koji Nakanishi*
Department of Chemistry, Columbia University New York, New York 10027 Received April 16, 1985 Mathematical second derivatization of absorption spectra has primarily been used as a tool for enhancing resolution or clarifying maxima in strongly scattering samples. However, the technique can be exploited to a much greater extent, especially when coupled with subtractive methods. This is shown by the usage of differential second-derivative UV (differential SEDUV) and FTIR (differential SEDIR) spectroscopy in the microgram-scale structure determination of complex molecules exemplified by the mitomycin C (MC, l)/guanine adducts 2 and 3. The method
MC
2 M-guanine A , l a -
4. .
3 M-guanine E ,
N
Ip-
described can be performed on most computerized UV and IR spectrometers without weighing of samples and should be generally applicable to a wide variety of problems. Treatment of M C with the dinucleoside phosphate d(GpC) under acidic conditions yields a complex mixture from which were isolated two major adducts M-guanines A 2 and B 3' (M denotes 0 1985 American Chemical Society
J . Am. Chem. SOC.,Vol. 107, No. 21, 1985 61 19
Communications to the Editor NI-Me
e
2-Nhfe
N3-Me
6-OEI
N7-CH-CH-OH
&Me
f
Figure 1. U V of guanines, 0.1 M K phosphate, p H 7.0. Arrows denote peak positions in nanometers; parenthesized numerals denote shoulders: (A-F) absorption spectra; (a-f) second-derivative (SEDUV) spectra. I f
U n
//
5
Figure 3. F T I R of guanines, Me,SO-d,. The three major characteristic frequencies are shown in reciprocal centimeters (A-F) and by asterisks (a-f): (A-F) absorbance spectra; (a-f) second derivative (SEDIR) spectra.
diff. deriv. (2-9
Figure 2. (A) U V of adduct 2 and mitosene 4 and differential spectrum of (2 - 4). (a) S E D U V of 2 and 4 and differential S E D U V of (2 - 4). (B) F T I R of adduct 2 and mitosene 5. (C) Differential F T I R of ( 2 5). (c) Differential S E D I R of (2 - 5). Asterisked peaks in (c) correspond to labeled peaks in (C).
mitosene2). 'H N M R clarified the structure of the mitosene moiety but gave very limited information on the guanine portion due to the lack of usable proton signals other than 8'-H. The usage of UV in determining the point of attachment of the mitosene to the guanine moiety was therefore explored. As shown in Figure 1, the UV spectra of alkylated guanines do differ according to the substitution site but are not sufficiently characteristic to serve as fingerprints, especially when subtraction of strongly absorbing chromophores are involved (see below). In contrast, the SEDUV spectra (Figure la-f)334 show greatly enhanced resolution as seen by clarification of shoulders (compare Figure 1, curves C/c, E/e, F/f) and resolution of overlapping bands into two peaks (Figure 1, curves C/c, E/e). Increase in derivative order leads to further enhanced resolution but also to reduced S/N and hence the second derivative is the best compromise in most cases. (1) Following paper in this issue. (2) "Mitosene" refers to the structure as in 4 without substituents in the I-, 2-, and 7-positions. Webb, J. S.; Cosulich, D. B.; Mowat, J. H.; Patrick, J. B.; Broschard, R. W.; Meyer, W. E.; Williams, R. P.;Wolf, C. F.; Fulmore, W.; Pidacks, C.; Lancaster, J . E. J . Am. Chem. SOC.1962, 84, 3185. (3) Cahill, J. E. Am. Lab. (Fuirfeld, Conn.) 1979, 1 1 , 79 and references cited therein. (4)Perkin-Elmer (PE) 320 UV spectrophotometer linked to a PE 3600 data station was used. Normal and SEDUV spectra were acquired in real time at 1-nm resolution in the absorbance mode. All SEDUV spectra were scanned at a AX value of 6 nm. Sample concentrations were adjusted to a maximum absorbance of 0.5 to avoid subtraction errors arising from Beers' law deviations. ( 5 ) If the spectra measured at pH 7 are not sufficiently different to be of diagnostic use, Figure 1 curves A / a vs. B/b, differentiation can be achieved by measurements at different pH's.
The absorption spectrum of M-guanine A adduct 2 (Figure 2A) is a summation of mitosene bands at 245 ( t 14400), 309 (lOOOO), 350 (3200), and 530 nm (700) and substituted guanine bands at 204-215 (e 15000), 234-246 (lOOOO), and 267-284 nm (8000). Computer-assisted subtraction of the spectrum of model mitosene 4 from adduct 2 using the mitosene 350-nm absorbance as the normalizing wavelength leads to two (2 - 4) difference spectra, Figure 2A in the normal mode and Figure 2a in the SEDUV mode. Comparison of the difference spectra with authentic sets Figure 1, curves A-F and a-f, shows they match the spectra of N7-(hydroxyethyl)guanine (Figure 1E/e), However, the agreement between the difference curve in Figure 2A with maxima at 206/284 nm and reference curve Figure 1E with maxima at 215/284 nm is not convincing. In contrast, agreement between the SEDUV set is excellent: 205/216/245/285 nm (Figure 2a) vs. 205/215/246/284 nm (Figure le). Analysis of spectra of adduct M-guanine B 3 leads to the same conclusion. The subtractive technique where difference curve (2 - 4) is compared with the spectra of reference guanines is valid since (i) the exciton coupling between two identical chromophores shifts the peaks maximally by 6 nm;6 in adducts 2/3 the shift is much less because the two chromophores are different, and (ii) the ionic conditions of measurements preclude the possibility of guanine peaks in adducts 2/3 being shifted by an intramolecular H bond. The l a - and 10-configurations were determined by the C D method, 2, 525 nm (& -0.081) and, 3, 520 nm ( A t +0.112).' We previously showed that differential FTIR using a lyophilized KBr technique offered a powerful method for determining sub* demonstrated below, stitution positions on purine b a s e ~ , ' ~ . As resolution is greatly enhanced and shoulders became clear bands when solution spectra taken in an attenuated total reflectance CIRCLE microcell' are recorded as second derivatives. Figure 3A-F shows regular absorbance spectra'" of substituted guanines whereas Figure 3a-f shows the corresponding second-derivative spectra. (6) Harada, N.: Uda, H . J . Chem. SOC.,Chem. Commun. 1982, 230. (7) (a) Tomasz, M.; Lipman, R.; Snyder, J. K.; Nakanishi, K. J . A m . Chem. SOC.1983, 105. 2059. (b) Tomasz, M.; .lung, M.; Verdine, G.; Kakanishi, K. J . Am. Chem. Soc. 1984, 106, 7367. (8) Kasai, H.; Nakanishi, K.; Traiman, S. J . Chem. Soc., Chem. Commun. 1978, 798. (9) Barnes Analytical Division, Spectra-Tech Inc., 652 Glenbrook Road. Stamford, C T 06906. (10) IBM IR-85 equipped with an MCT detector, 2-cm-' resolution, in MeiSO-d,, using an A T R C I R C L E microcel19 fitted with a ZnSe rod, and corrected for solvent and H z O vapor. Each spectrum is the Fourier transform of 2500 accumulations. SEDIR spectra were calculated on the digitized absorbance files using a smoothing factor of -7.
6120
J . Am. Chem. SOC.1985, 107, 6120-6121
The 1800-1400 cm-' region FTIR of adduct 2 (Figure 2B, curve 2) consists of bands due to the mitosene and guanine moieties. The mitosenes exemplified by 5 exhibit a ca. 1722-cm-I band due to the carbamate group (Figure 2B, curve 5 ) but this region is completely transparent in guanines. Weighted subtraction of the spectrum of 5 from that of 2 using the 1722-cm-' band as subtraction marker thus produces the difference spectrum Figure 2C which is composed of guanine-related bands. The peaks in curve C of Figure 2 compare most closely with the FTIR 3 N7-(hydroxyethyl)guanine," curve E, Figure 3, but the conclusion is not unambiguous. In contrast, a comparison of differential SEDIR curve in Figure 2c with curves in Figure 3a-f shows clearly that the three asterisked peaks together with a few others in curve c in Figure 2 only match those of Figure 3e. The above-mentioned examples demonstrate that diff. SEDUV and differential SEDIR offer extremely powerful micromethods for structural studies of molecules containing two (or more) nonconjugated moieties. While the present example is rather specific, we believe that the additional information gained from second derivatization should make it an attractive technique in a broad range of investigations.
Acknowledgment. We are grateful to Prof. M. Tomasz, Hunter College, CUNY, New York, for a supply of samples and discussions, Dr. Reiji Takeda, Suntory Foundation for Bioorganic Research, Osaka, for discussions, and N I H Grant G M 34509. Supplementary Material Available: Expanded scale FTIR and SEDIR spectra for all compounds; FTIR and SEDIR spectra of M-guanine B (9 pages). Ordering information is given on any current masthead page. (11) Brookes, P.; Lawley, P. D. J . Chem. SOC.,Chem. Commun. 1961, 3923.
Nature of the Destruction of Deoxyguanosine Residues by Mitomycin C Activated by Mild Acid pH Maria Tomasz,*+ Roselyn Lipman,+ Gregory L. Verdine,t and Koji Nakanishi*i
Department of Chemistry, Hunter College City University of New York New York, New York 10021 Department of Chemistry, Columbia University New York, New York 10027 Received April 16, 1985 Alkylation of genomic deoxyguanosine residues at the 7-position has been implicated as the primary event in the chemically induced carcinog .nesis/mutagenesis resulting from exposure to agents such as aflatoxin B1,'.* N-mustards, and dimethyl sulfate.' The modified deoxyguanosine residues produced by 7-alkylation decompose readily via (i) loss of the ribofuranoside moiety to form apurinic DNA sites or (ii) imidazdium-ring scission to generate N-formamidopyrimidine (NFP) DNA bases (see below). With respect to the latter case, while numerous studies have been conducted on NFP-ribosides, including DNA, their exact structures have continued to elude definition.'-2 We wish to report that mitomycin C 3 (MC, l), the clinically used antitumor antibiotic, alkylates ~ ( G P C and ) ~ D N A in vitro 'City University of New York. Columbia University. ( I ) Singer, B.; Grunberger, D. "Molecular Biology of Mutagens and Carcinogens"; Plenum Press: New York, 1983. (2) Croy, R. G.; Essigmann, J. M.; Reinhold, V. N.; Wogan, G. N. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 1745. (3) Absolute configuration: Hirayama, N.; Shirahata, K. J . Am. Chem. SOC.1983,105,7199. Hornemann, U.; Heins, M. J. J . Org.Chem. 1985, 50, 1301. Verdine, G.; Nakanishi, K. J . Chem. Soc., Chem. Commun., in press. (4) This dinucleoside phosphate was chosen as the simplest model for a polynucleotide inasmuch as M C binding to DNA increases with increasing G-C base-pair content.
Figure 1. Products resulting from MC (1) and d(GpC) (3). Intermediate 17 has not been isolated. M in 4-8 denotes the mitosene moiety.
at the guanine 7-position under acidic a ~ t i v a t i o n . This ~ finding raises the possibility that M C activation in vivo may well be accomplished by protonation,6 the other possibility being the well-known (in vitro) reductive activation of MC.'+ Furthermore, we report the full structures of the elusive imidazolium-cleaved N F P moieties generated from base-treated 7-ethylguanosine (9) (Figure 2). On the basis of the latter model studies, we have also characterized the NFP-deoxyribose-mitosene 6-8 system shown in Figure 1. An aqueous solution of 18 pmol of d(GpC) (3) acidified to pH 3.5-4.0 with Bio-Rad AG 50-W-X-B beads was treated with MC (20 pmol) and the solution adjusted to 2.5 mL with water and maintained at pH 3.5-4.0 by additions of 0.01 N HCI. The solution was neutralized after 3.5 h, room temperature, and chromatographed on Sephadex C1-25~to give adduct fractions designated M-guanines and M-d(GpC); digestion of M t l ( G p C ) with snake venom diesterase gave M-dG, (major) and M-dGII (minor). The latter, the major product under reductive conditions, consists of type 2 mitosenelo l a - and lp-adducts to the O6of dG.9 The M-guanines were separated by HPLC, Ultrasphere ODS, MeCN/0.02 M aqueous K H 2 P 0 4(pH 5.0), 8/92, into 4 and 5 (Figure 1). The ' H N M R in Me2SO-d6 showed three exchangeable 2 H signals assigned as follows by comparison with 2P,7-diamino- la-hydroxymitosene and guanine: 6.74/6.58 (4) and 6.52/6.36 ppm ( 5 ) , loa-/7-NH,; 6.20 (4) and 6.18 ppm ( 5 ) , guanine 2-NH2 (mitosene 2-NH2 signals are broad and rarely observed). The guanine 8-H's were observed at 7.91 (4) and 7.46 ppm ( 5 ) . The rest of the spectra showed typical mitosene peaks;9 4 and 5 are thus type 2 adducts. The linkage of the mitosene group to N-7 was determined by differential second-derivative UV and FTIR spectroscopy." Finally, the CD of 4, A6 -0.081 at 525 nm, and 5, A€ +0.112 at 520 nm show them to be l a - and lpadducts, respe~tively.~ HPLC (conditions same as above) of 200 pg of M-dGI gave an ill-resolved four-peak pattern which was reproduced upon reinjection of any single peak; M-dGI is therefore a mixture of interconverting compounds. The ' H N M R showed nonex(5) Lipman, R.; Tomasz, M. J . Am. Chem. Soc. 1979, 101, 6063. (6) The weakly acidic conditions used here for activation may mimic the low pH found inside gastric and solid tumors, for which M C is an effective treatment. Douglass, H. 0.;Lavin, P. T.; Goudsmit, A,; Klassen, D. J.; Paul, A. R. J . Clin. Oncol. 1984, 2, 1372. (7) Tomasz, M.; Lipman, R. Biochemistry 1981, 20, 5056. (8) Hashimoto, Y.; Shudo, K.; Okamoto, T. Tetrahedron Lett. 1982, 677; Chem. Pharm. Bull. 1983, 31, 861; Acc. Chem. Res. 1984, 17, 403. (9) Tomasz, M.; Lipman, R.; Snyder, J . K.; Nakanishi, K. J . Am. Chem. Soc. 1983, 105, 2059. Tomasz, M.; Jung, M.; Verdine, G.L.; Nakanishi, K. J . Am. Chem. SOC.1984, 106, 7367. (1 0) Mitosene derivatives are the end-products of M C activation/alkylation. "Mitosene" refers to the structure as in 2, without substituents at C-1,-2,-7: Webb, J. S . ; Cosulich, D. B.; Mowat, J. H.; Patrick, J. B.; Broschard, R. W.; Meyer, W. E.; Williams, R. P.; Wolf, C. F.; Fulmore, W.; Pidacks, C.; Lancaster, J. E. J . Am. Chem. SOC.1962, 84, 3185. (1 1) Verdine, G. L.; Nakanishi, K., preceding paper in this issue.
0002-7863/85/ 1507-6120$01.50/0 0 1985 American Chemical Society
